The proteolytic activity of a 700kDa complex called the 20S proteasome is essential for all eukaryotic life. Isolated proteasomes are inactive because entrance gates to the internal catalytic chamber are by default in the closed position. Substrates gain access to the catalytic centers (and products leave) via activator complexes that bind to the proteasome ends and open the entrance gate. During the previous funding period our results included determining the crystal structure of a 1.1 MDa, 42 sub-unit complex of 20S proteasome with an activator of the 11S family. This structure provided considerable insight into proteasome mechanism, suggests a model for how some 11S activators promote production of ligands for MHC class I molecules, and provides the foundation for our current specific aims. Aims 1 and 2 propose biochemical and structural studies that follow directly from the published 20S/11S structure. Aim 1 will test the hypothesis that the specific open gate conformation seen in the 20S/11S structure is also adopted in other proteasome/activator complexes. Because the structure already determined is for a highly diverged non-cognate complex, aim 2 will determine how cognate mammalian 20S/11S complexes are formed and activated. Aim 3 is to determine structural basis for the activation of proteasomes by peptides. Aims 4 and 5 extend the structural studies to the two other known types of proteasome activator, PA200 and 19S, respectively. PA200 is a 250 kDa protein that functions in DNA repair (perhaps by recruiting proteasome), and the 19S activator is a 900 kDa complex of 18 different protein subunits that delivers most of the physiological substrates for proteasomal degradation. Each of these aims presents obvious challenges. However, expression and purification systems have been developed that justify sustained effort towards these aims.